BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development

Abstract

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x L .To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.

Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function

“…After 48 h expression of Beclin-1-GFP and the targeted Bcl-2 constructs (mit-Bcl-2 and ER-Bcl-2) or non-targeted wt-Bcl-2, cells were stained for Bcl-2 expression (Figure 1a). Without overexpression of Bcl-2, ectopically expressed Beclin-1-GFP was distributed evenly throughout the cytoplasm, consistent with recent data examining the distribution of Beclin-1-GFP in a transgenic mouse model (Arsov et al, 2008). However, upon cotransfection with the Bcl-2 variants, the pattern changed, being uniquely organellar with mit-or ER-Bcl-2 and intermediate between the two with wt-Bcl-2.…”

Autophagy-active beclin-1 correlates with favourable clinical outcome in non-Hodgkin lymphomas

“…10,33,34 In this study, we considered the role of beclin-1 as an autophagy regulator, because this process has been shown to have a crucial role in lymphatic tissue homeostasis. 6,7,27 On formation of the autophagosome, the microtubule-associated protein LC3 (isoform I) is modified to generate the truncated and lipidated isoform LC3-II, that is eventually inserted into the autophagosome membrane. 14 Thus, autophagic vacuoles can be easily and reliably detected based on LC3 staining.…”

“…25,26 The expression level of beclin-1 changes during T and B lymphocyte maturation and T-cell activation. 27 Fluctuation of beclin-1 expression was shown to parallel that of bcl-2 during the maturation process of T cells in the thymus and of B cells in bone marrow. 27 In addition, overexpression of ectopic beclin-1 in thymocytes exacerbates glucocorticoid-induced apoptosis.…”

“…27 Fluctuation of beclin-1 expression was shown to parallel that of bcl-2 during the maturation process of T cells in the thymus and of B cells in bone marrow. 27 In addition, overexpression of ectopic beclin-1 in thymocytes exacerbates glucocorticoid-induced apoptosis. 27 Altogether these data imply that autophagy has a critical role in lymphocyte homeostasis.…”

“…27 In addition, overexpression of ectopic beclin-1 in thymocytes exacerbates glucocorticoid-induced apoptosis. 27 Altogether these data imply that autophagy has a critical role in lymphocyte homeostasis. Consistent with such a role, transgenic mice lacking one beclin-1 allele present with a reduced autophagy activity in the lymph node germinal centre and show a high incidence of spontaneous lymphomas of the B lineage.…”